De novo methylation of CpG islands, which are generally not methylated on autosomal genes in normal cells, is one of the most common genomic alterations in human cancers. Focal hypermethylation occurs in parallel with a genome-wide demethylation during carcinogenesis. The aberrant methylation of CpG islands, if it occurs in promoter regions, contributes to the inactivation of tumor suppressor, growth regulatory and other cancer related genes. Epigenetic modification of CpG islands is now considered to be a significant contributor to gene inactivation during carcinogenesis. The objectives of this proposal are to take advantage of new technologies developed for the analysis of chromatin structural changes, quantitative DNA methylation analysis and siRNA technology to determine the underlying mechanisms responsible for altered methylation patterns in human cancer. Additionally, we will continue the characterization of a drug we have discovered (Zebularine) which shows promise as a selective inhibitor of DNA methylation in cancer but not in normal cells. We will focus our attention on four specific aims. Firstly, we will use a new inducible siRNA vector we have developed for the production of conditional gene knockouts to determine the effects of DNA methyltransferase enzymes and chromatin modifying enzymes on the rate of remethylation of CpG islands following a transient demethylation induced by DNA methylase inhibitors. Secondly, we will utilize a new fingerprinting technique we have developed to determine how histone modification including acetylation and methylation become differentially distributed when the genome reacts to an induced demethylation. Thirdly, we will study the role of methylation in potentially silencing two genes at one time in the bi-directional CpG island promoter associated with the hMLH1 gene. Finally, we will continue to define the mechanisms of action of zebularine, which appears to be preferentially incorporated into the DNA of cancer relative to normal cells. Once in DNA, it functions as a mechanism-based inhibitor of DNA methyltransferase and appears to show specificity for the DNMT1 enzyme. This drug is orally active and might possibly find use as a chemotherapeutic agent.